Butoconazole

The in vitro susceptibility tests by using the CLSI reference broth microdilution method were performed for all species isolates number less than 100 strains. C. albicans and C. glabrata were randomly selected for the test. Those include 1272 C. albicans strains (including 998 isolates from VVC and 274 from RVVC) and 267 C. glabrata strains(including 197 isolates from VVC and 70 from RVVC). The MIC of Candida for all agents was read following 24–48 h incubation. The antifungals used were amphotericin B (Sigma, USA), Anidulafungin(Selleckchem, USA), Butoconazole(Sigma, USA), Caspofungin(Sigma, USA), Clotrimazole(Sigma, USA), Fluconazole(Sigma, USA), Flucytosine(Sigma, USA), Itraconazole(Sigma, USA), Micafungin(Selleckchem),Miconazole(Sigma, USA), Nystatin (Amresco, USA), Terbinafine(Santa Cruz, USA),Terconazole(Sigma),and Voriconazole(Fluka, USA). Quality control was performed as recommended in CLSI documents M27-A3 and M60 by using ATCC 90028 which is a reference strain of C. albicans and all results of the control were within established ranges [20 , 21 ].

Itraconazole, miconazole, fluconazole, butoconazole, and caspofungin were obtained from Sigma-Aldrich (St. Louis, MO, USA). The in-vitro activity of six antifungal drugs was assessed by means of the Clinical and Laboratory Standards Institute (CLSI) M-60 protocol [ 17
]. The minimal inhibitory concentration (MIC) of antifungals was determined after 24 h. Candida parapsilosis ATCC 22019 was chosen as a control in every run. The results were analyzed using the CLSI M60 guideline.

Bifonazole, butoconazole, clotrimazole, econazole, itraconazole, ketoconazole, miconazole, posaconazole, ravuconazole, terconazole, tioconazole, voriconazole and benzyl-4-hydroxybenzoate (Internal Standard, IS) (>98% purity grade), sodium phosphate monobasic and sodium phosphate dibasic (>99% purity grade), phosphoric acid (90% purity grade) and trichloroacetic acid (>99% purity grade) were purchased from Sigma-Aldrich (Milan, Italy).
The commercial drugs were obtained from a local Pharmacy after medical prescription. Both HPLC-grade Acetonitrile (AcN) and methanol (MeOH) were supplied by Carlo Erba (Milan, Italy) and were used without further purification. The water for HPLC analysis was generated by using a Millipore Milli-Q Plus water treatment system (Millipore Bedford Corp., Bedford, MA). Oasis HLB (1 mL, 30 mg) and Sep-Pak (1 mL, 50 mg) were obtained from Waters (Milford, MA), while Strata-X (1 mL, 30 mg) was purchased from Phenomenex (Torrance, CA). MEPS device (syringe) and replacement needle with C₁₈ stationary phase were purchased from SGE Analytical Science (Melbourne, Australia).